反応 #44519

反応方程式

Tris-HCl

MgCl2

nicotinamide

PRPP

→

nicotinamide mononucleotide

nicotinamide

PRPP

反応条件

後処理

1. 1
   その他Nampt reaction products
2. 2
   その他The Nampt reaction
3. 3
   その他The reaction was terminated
4. 4
   workup.ADDITIONby adding 125 μl of 1 M HClO4
5. 5
   その他Protein was then precipitated at 18,000 g, and 500 μl of the supernatant
6. 6
   workup.ADDITIONAfter centrifugation, 100 μl of sample was mixed with 400 μl of Buffer A (50 mM K2PO4/KHPO4, pH 7.0)
7. 7
   その他The products from Nampt reaction
8. 8
   その他Results of HPLC detection of Nampt reaction products

実験手順

High performance liquid chromatography was used to detect Nampt reaction products. HPLC was performed with Waters 515 pumps and a 2487 detector (Waters, Mass.) with a Supelco LC-18-T column (15 cm×4.6 cm; Supelco, Pa.). The Nampt reaction was conducted at 37° C. for 15 min in 500 μl of reaction buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 50 mM nicotinamide, 0.2 mM PRPP) with 50 μg of the recombinant Nampt protein. The reaction was terminated by adding 125 μl of 1 M HClO4. Protein was then precipitated at 18,000 g, and 500 μl of the supernatant was neutralized with 40 μl of 3 M K2CO3. After centrifugation, 100 μl of sample was mixed with 400 μl of Buffer A (50 mM K2PO4/KHPO4, pH 7.0) and loaded into the HPLC system. The products from Nampt reaction were monitored by absorbance at 261 nm. Results of HPLC detection of Nampt reaction products showed that the mouse Nampt produced nicotinamide mononucleotide (NMN) from nicotinamide and PRPP (see, e.g., FIG. 4D). Nampt failed to catalyze the synthesis of nicotinic acid mononucleotide (NaMN) from nicotinic acid and PRPP (see, e.g., FIGS. 13A and 13B), confirming the substrate specificity of this enzyme. In isolated reactions, it was also confirmed that Nmnat catalyzed the synthesis of NAD from NMN and ATP.

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